Compounds for the treatment of AIDS and other viral diseases

ABSTRACT

The present invention provides methods for treating Acquired Immunodeficiency Syndrome (AIDS) and other viral diseases and Human Immunodeficiency Virus (HIV) related infections by administering one or more compounds of formula I: 
                         
wherein:
         the dotted line represents a single or a double bond; and   R 1  and R 2  are the same or different and independently of each other represent —CH 2 OH, —CH 2 OR 4 , —CH(OH)CH 3 , —CH(OR 4 )CH 3  or a group represented by the formula:       
                         
or salts or hydrates thereof in a carrier which minimizes micellar formation or van der Waals attraction of molecules of said compound. The invention also provides S enantiomeric forms of such compounds which possess the ability to inhibit cell growth whilst being of low toxicity to such cells and methods of making such compounds.

RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 11/089,157, filed Mar. 24, 2005, which claims the benefit ofpriority from U.S. provisional patent application Ser. No. 60/557,087,filed Mar. 26, 2004; both of which are hereby incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to compounds for use in the treatment ofAIDS and other viral diseases and HIV+ related infections andcompositions containing such compounds. The present invention alsoprovides methods for the treatment of such diseases and infections andmethods of making such compounds and compositions.

BACKGROUND OF THE INVENTION

Diseases of the immune systems pose a major threat owing to thepotentially devastating effects that such diseases can have on humanity.An example of a disease of the immune system is AcquiredImmunodeficiency Syndrome (AIDS). A retrovirus designated humanimmunodeficiency virus (HIV) is the etiological agent of AIDS, a complexdisease that includes the progressive destruction of the immune systemand degeneration of the central and peripheral nervous system. AIDS isone of the deadliest diseases to have struck humans in recent times, andit has reached epidemic proportions. It is estimated that over eighteenmillion people are infected with HIV worldwide. AIDS has been reportedin more than one hundred and twenty-three countries.

Currently, a number of drugs and drug combinations are available totreat and control AIDS. There is an ongoing search in to identify potentcompounds that are effective against AIDS and HIV+ related infections.Representative examples of methods and compounds for treating andcontrolling AIDS are disclosed in, e.g., U.S. Pat. Nos. 6,180,634;6,120,772; 6,040,434; 6,015,796; 5,905,077; 5,888,511; 5,846,978;5,811,462; 5,747,540; 5,744,906; 5,631,088; 5,504,065; 5,491,166;5,475,136; 5,430,064; 5,413,999; 5,229,368; 5,162,499; 5,108,993;5,059,592 and 5,028,995.

Diseases of the immune system pose a major problem to society.Epidemiological statistics relating to AIDS and other viral diseasesshow an ever increasing prevalence of such diseases, with global andregional health organizations predicting catastrophic consequences on amass scale unless effective and easily applicable means are provided andimplemented for the control of such diseases.

Accordingly there is a need in the art to develop potent compounds thatare effective in the treatment, prevention and control of AIDS and otherviral diseases and HIV+ related infections.

SUMMARY OF THE INVENTION

The present invention provides improvements in or relating to compoundsfor use in the treatment of AIDS and other viral diseases and HIV+related infections and the like and compositions comprising suchcompounds. The present invention also provides methods for making suchcompounds and compositions and methods of treating or controlling suchdiseases or infections.

According to one aspect of the present invention therefore there isprovided a method of treating, preventing or controlling a viraldisorder by administering to a patient in need thereof a compoundrepresented by the structure of formula I:

-   -   wherein:    -   the dotted line represents a single or a double bond.

In some embodiments, R₁ and R₂ may be the same or different andindependently of each other may represent —CH₂OH, —CH₂OR₄, —CH(OH)CH₃,—CH(OR₄)CH₃ or a group represented by the formula:

-   -   where R₄ is a linear or branched C₁-C₄ alkyl; R₅ is H, OH or OR₆        (where R₆ is a linear or branched C₁-C₄ alkyl); and    -   A-B is a group represented by the formula:

In some embodiments, m may be an integer of 0 or 1, n may be an integerof 1-500, and X may be O, —CH₂O, —CH₂CH₂O, —CH(CH₃)CH₂O or —CH₂CH(CH₃)O.

Alternatively, m may be 1, n may be an integer of 0 to 500, and X may be—CH₂O, —CH₂CH₂O, —CH(CH₃)CH₂O or —CH₂CH(CH₃)O.

Z may be —CH₂CH₂O, —CH(CH₃)CH₂O or —CH₂CH(CH₃)O.

In some embodiments, n may be an integer of from 1-200, particularly1-100.

Thus, in particular, X may be O, —CH₂O, —CH₂CH₂O, —CH(CH₃)CH₂O or—CH₂CH(CH₃)O, Z may be —CH₂CH₂O, —CH(CH₃)CH₂O or —CH₂CH(CH₃)O, m may be0 or 1 and n may be 0-50, but preferably m and n may not both be 0. Morepreferably, n may be an integer of from 1-50.

In some embodiments, n may be an integer of from 5-75. For example, nmay be 5, 6, 7, 8, 10, 11, 12, 13, 15, 16, 17, 18, 20, 25, 30, 33, 34,35, 40,45, 50, 60, 65, 68, 69, 70, or 75. Preferably, n is 7, 12, 17, 34or 69.

In some embodiments, R₁ may be —CH₂OH, —CH₂OR₄, —CH(OH)CH₃ or—CH(OR₄)CH₃.

Alternatively, R₁ may be:

-   -   wherein R₅ is H or OH. Thus, in some embodiments, R₁ may be        phenyl or:

In some embodiments, R₂ may be —CH₂OH, —CH₂OR₄, —CH(OH)CH₃ or—H(OR₄)CH₃.

Alternatively, R₂ may be:

-   -   wherein R₅ is H or OH. Thus, in some embodiments, R₂ may be        phenyl or

In a further aspect of the present invention there is therefore provideda method of treating, preventing or controlling a viral disorder byadministering to a patient in need thereof a compound represented by thestructure of formula II:

-   -   wherein:    -   the dotted line represents a single or a double bond; and    -   R₅ and R₅′, independently of each other, are H, OH or OR₆ (where        R₆ is a linear or branched C₁-C₄ alkyl).

As before, m may be an integer of 0 or 1, n may be an integer of 1-500,and X may be O, —CH₂O, —CH₂CH₂O, —CH(CH₃)CH₂O or —CH₂CH(CH₃)O.

Or m may be 1, n may be an integer of 0 to 500, and X may be —CH₂O,—CH₂CH₂O, —CH(CH₃)CH₂O or —CH₂CH(CH₃)O.

Z may be —CH₂CH₂O, —CH(CH₃)CH₂O or —CH₂CH(CH₃)O.

In some embodiments, n may be an integer of from 1-200, particularly1-100.

Thus, in particular, X may be O, —CH₂O, —CH₂CH₂O, —CH(CH₃)CH₂O or—CH₂CH(CH₃)O, Z may be —CH₂CH₂O, —CH(CH₃)CH₂O or —CH₂CH(CH₃)O, m may be0 or 1 and n may be 0-50, but preferably m and n may not both be 0. Morepreferably, n may be an integer of from 1-50.

In some embodiments, n may be an integer of from 5-75. For example, nmay be 5, 6, 7, 8, 10, 11, 12, 13, 15, 16, 17, 18, 20, 25, 30, 33, 34,35, 40, 45, 50, 60, 65, 68, 69, 70, or 75. Preferably, n is 7, 12, 17,34 or 69.

In some embodiments, m may be 0. Alternatively, X may be —CH₂O, and mmay be 1.

Thus, the present invention comprehends methods of treating, preventingor controlling a viral disorder by administering to a patient in needthereof a compound represented by the structure of formula III:

-   -   wherein the dotted line, R₅, R₅′, Z and n have the same meanings        as recited above in relation to formula I and II.

In some embodiments, Z may be —CH(CH₃)CH₂O, and accordingly the presentinvention embraces methods of treating, preventing or controlling aviral disorder by administering to a patient in need thereof a compoundrepresented by the structure of formula IV:

-   -   wherein the dotted line, R₅, R₅′ and n have the meanings as        ascribed to them above in relation to formula I and II.

In some embodiments, R₅ is H. In some embodiments, R₅ is OH.

In some embodiments, R₅′ is H. In some embodiments, R₅′ is OH.

In some embodiments, n is an integer of 1-20. In some embodiments, n isan integer of 10-20. In some embodiments, n is 17. Alternatively, n maybe an integer of 1-10, preferably 5-10, e.g. n=7.

The present invention also includes salts or hydrates of the compoundsrepresented by the structures of formula I, II, III and IV.

In a particular aspect of the present invention, there is provided amethod of treating, preventing or controlling a viral disorder byadministering to a patient in need thereof a compound of formula A:

-   -   wherein R is a polyalkylene glycol polymer having p units, where        p is an integer from 1-100.

In some embodiments, the polyalkylene glycol polymer may bepolyisopropylene glycol.

p may be an integer of from 5-75. For example, p can be 5, 6, 7, 8, 10,11, 12, 13, 15, 16, 17, 18, 20, 25, 30, 33, 34, 35, 40, 45, 50, 60, 65,68, 69, 70 or 75. Preferably, p is 7, 12, 17, 34 or 69.

Further, the present invention provides methods of treating, preventingor controlling a viral disorder by administering to a patient in needthereof compounds of formulae B, C, D, E or F:

-   -   wherein R and p have the same respective meanings as given in        relation to formula A.

In a different aspect of the present invention there is provided acomposition for treating, preventing or controlling a viral disordercomprising one or more compounds of formulae I, II, III, IV, A, B, C, D,E or F. Thus, in some embodiments, the present invention provides apharmaceutical composition for treating, preventing or controlling aviral disorder comprising as an active ingredient one or more compoundsof formula I, II, III, IV, A, B, C, D, E or F, together with one or morepharmaceutically acceptable excipients or adjuvants. In someembodiments, the pharmaceutical composition of the invention maycomprise one or more compounds of formulae I or II.

In another aspect of the present invention there is provided a methodfor the treatment, prevention or control of AIDS and other viraldiseases and HIV+ related infections, which method comprisesadministering one or more compounds of formula I, II, III, IV, A, B, C,D, E or F and/or a pharmaceutical composition comprising one or morecompounds of formula I, II, III, IV, A, B, C, D, E or F to a patient inneed thereof. Typically, one or more compounds of formula I or II may beused.

In yet another aspect, the present invention comprehends the use of oneor more compounds of formula I, II, III, IV, A, B, C, D, E or F ashereinbefore defined in the manufacture of a medicament for thetreatment, prevention or control of AIDS and other viral diseases andHIV+ related infections.

In yet another aspect of the present invention there are providedmethods for inducing AICD, inducing apoptosis, inhibiting a chemokinereceptor, inhibiting malignant metastasis or inhibiting fibrosis oraberrant fibroblast proliferation, which methods each compriseadministering one or more compounds of formula I, II, III, IV, A, B, C,D, E or F and/or a pharmaceutical composition comprising one or morecompounds of formula I, II, III, IV, A, B, C, D, E or F to a patient inneed thereof. In some embodiments, one or more compounds of formulae Ior II are used. Said compounds of the invention may suitably beadministered in a carrier which minimises micellar formation or van derWaals attraction of molecules of the compounds; an example of such acarrier is DMSO.

In some embodiments, the compounds of the present invention may excludeN-cinnamoyl-D,L-phenylalaninol,N-[1-hydroxymethyl-2-(1H-indol-3-yl)-ethyl]-3-phenyl-propionamide,N-[1-hydroxymethyl-2-phenyl-ethyl]-3-(4-hydroxy-phenyl)-propionamide,N-(1-hydroxymethyl-2-phenyl-ethyl)-3-(1H-indol-3-yl)-propionamide,N-(1-hydroxymethyl-2-phenyl-ethyl) -3-phenyl-propionamide,N-[1-hydroxymethyl-2-(4-hydroxyphenyl)-ethyl]-3-phenyl-propionamide, orN-[1-hydroxymethyl-2-(1H-imidazol-4-yl)-ethyl]-3-(4-hydroxyphenyl)-propionamide.

In accordance with a different aspect of the present invention, theS-enantiomeric forms of the compounds of formula A to F may beparticularly advantageous in that embodiments thereof have been found toexhibit useful cell division inhibitory properties whilst at the sametime demonstrating little or no toxicity to animal cells at theconcentration levels required to achieve such cell inhibition.

Accordingly, in a particular aspect of the present invention there isprovided a compound of formula A′, B′, C′, D′, E′ or F′ as follows:

-   -   wherein the chiral centre indicated by * is in the        S-enantiomeric conformation, and R represents a polyalkylene        glycol polymer having p units, in which p is an integer from        1-100. Preferably R is polyethylene glycol or polypropylene        glycol and p is an integer in the range 1 to 20. Particularly        preferred are compounds where p is 7 or 17.

In a particular aspect of the present invention are provided (S)2-N(3-O-polypropyleneglycol)propylbenzene)-3-(4-hydroxyphenyl)propylamide (AV 61S, p=7) and(S)2-N(3-O-(polypropyleneglycol)-1-propyl-4-hydroxybenzene)-3-phenylpropylamide(AV 74S, p=7).

Said compounds of formula A′, B′, C′, D′, E′ or F′ as hereinbeforedefined may be used in methods of treatment of the human or animal bodyby therapy, for example for the treatment, prevention or control of AIDSand other viral diseases and HIV+ related infections as described aboveor for the treatment or prophylaxis of immuno-allergical or autoimmunediseases or for the treatment, prevention or control of organ or tissuetransplantation rejection in humans or animals as described in copendingPCT/IB2003/04993, the contents of which are incorporated herein byreference.

In a different aspect of the present invention is provided a method formaking a compound of formula A′, D′ or E′ as defined above whichcomprises:

-   -   (i) providing a compound of formula V:

-   -   wherein the chiral centre indicated by * is the S-enantiomer,        said compound of formula V comprising at least one hydroxy        phenyl group;    -   (ii) reacting said compound of formula V with a protecting agent        adapted to protect the phenylic hydroxyl group(s) on said        compound;    -   (iii) forming an alkali metal salt of the alkyl hydroxyl group;    -   (iv) reacting said alkali metal salt with a polyalkylene glycol        comprising a leaving group; and    -   (v) thereafter deprotecting said phenylic hydroxyl group(s) to        obtain said compound of formula A′, D′ or E′.

Preferably said compound of formula V comprises a phenyl group and anhydroxy phenyl group, such as a 4-hydroxy phenyl group.

Said polyalkylene glycol may be polyethylene glycol or polypropyleneglycol.

A preferred protecting agent in step (ii) is di-tert-butyl dicarbonate,but any other protecting group known to those skilled in the art for usein peptide synthesis may be used.

The alkali metal salt formed in step (iii) may be the potassium orsodium salt which may be obtained, for example, by reacting the(protected) compound of formula V with sodium or potassium ethoxide.

A preferred leaving group in step (iv) is mesyl (methane sulfonyl), butother suitable leaving groups are known to those skilled in the art.

Said compound of formula V may be formed by coupling a compound offormula X:

-   -   with a compound of formula Y:

-   -   wherein at least one of X and Y comprises an hydroxy phenyl        group.

In some embodiments, N-hydroxybenzotriazole (HOBt) anddicyclohexylcarbodiimide (DCC) may be employed as coupling agents, butother suitable coupling agents are known and available to those skilledin the art of peptide synthesis.

Said compound of formula X may be selected from3-(4-hydroxyphenyl)-propionic acid and hydrocinnamic acid. Said compoundof formula Y may be selected from L-tyrosinol hydrochloride and3-(4-hydroxyphenyl)-propionic acid.

It has been found that the method for making a compound of formula A′,D′ or E′ in accordance with the present invention gives surprisinglyhigh yields as compared with other possible methods such, for example,as those described in copending PCT/IB2003/004993. In particular, theyield of said compound of formula A′, D′ or E′ may be at least 20% wt.or 30% wt., with the majority of any other product(s) or residue beingcomposed substantially of unreacted polyalkylene oxide. In someembodiments, yields of 40% wt. or more may be obtained. Generally it hasbeen discovered that the yield of the desired compound may be greaterfor lower values of p. A particularly preferred value of p is 7. Anotherpreferred value is 17.

In accordance with yet another aspect of the present invention thereforethere is provided a composition comprising at least 20% wt. of acompound of formula A′, D′ or E′, wherein R and p are as defined above.Said composition may further comprise unreacted polyalkylene glycol as aside product. In some embodiments, said composition may comprise morethan about 30% wt. or 40% wt. of said compound, preferably more than 50%wt., and more preferably more than 75% wt., e.g. about 80% wt. or about85% wt.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below by way of example only. All publications,patent applications, patents and other references mentioned herein areincorporated herein in their entirety by reference. In the case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

The above description sets forth rather broadly important features ofthe present invention in order that the detailed description thereofthat follows may be better understood and that the present contributionsto the art may be better appreciated. Other objects and features of thepresent invention will be apparent from the following detaileddescription considered in conjunction with the accompanying drawings. Itis to be understood that the drawings are designed solely for thepurposes of illustration and not as a definition of the limits of theinvention, for which reference should be made to the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-15 illustrate the data obtained from the experiments describedin Examples 5-13 below.

DETAILED DESCRIPTION OF THE INVENTION

As contemplated herein, an “alkyl” group refers to a saturated aliphatichydrocarbon, including straight-chain, branched-chain and cyclic alkylgroups. In some embodiments of the present invention, the alkyl groupmay have 1-4 carbons. For example, the alkyl group may be a methylgroup. Alternatively, the alkyl group may be an ethyl group.Alternatively, the alkyl group may be a propyl group. Alternatively, thealkyl group may be a butyl group. The alkyl group may be unsubstitutedor substituted by one or more groups selected from halogen, hydroxy,alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino,alkylamino, dialkylamino, carboxyl, thio and thioalkyl.

Synthetic methodologies for obtaining the compounds of the presentinvention are disclosed in detail in the Examples section below.However, it should be apparent to those skilled in the art that thecompounds of the present invention may be prepared by any feasiblesynthetic method and that, except where stated otherwise, the synthesesset forth in the Experimental Details Section are in no way limiting.Compounds of the invention may be further modified as allowed by therules of chemistry. Such modifications include the addition of varioussubstituents (e.g., hydroxylation, carboxylation, methylation, etc.),generation of enantiomers, creation of acid- or base-addition salts orthe like. Other modifications include adding polyalkylene glycolpolymers.

In accordance with the present invention the compounds of the inventionmay be synthesised as polyalkylene glycol (PAG) conjugates. Typicalpolymers used for conjugation include poly(ethylene glycol) (PEG)—alsoknown as or poly(ethylene oxide) (PEO)—and polypropylene glycol(including poly isopropylene glycol). Such conjugates may be used toenhance solubility and stability and to prolong the blood circulationhalf-life of molecules.

In its most common form, a polyalkylene glycol (PAG), such as PEG, is alinear polymer terminated at each end with hydroxyl groups:HO—CH₂CH₂O—(CH₂CH₂O)_(q)—CH₂CH₂—OH.

The above polymer, alpha-, omega-dihydroxylpoly(ethylene glycol), canalso be represented as HO-PEG-OH, where it is understood that the-PEG-symbol represents the following structural unit:—CH₂CH₂O—(CH₂CH₂O)_(q)—CH₂CH₂—

-   -   where q typically ranges from about 4 to about 10,000. PEG is        commonly used as methoxy-PEG-OH, or mPEG, in which one terminus        is the relatively inert methoxy group, while the other terminus        is a hydroxyl group that is subject to ready chemical        modification. Additionally, random or block copolymers of        different alkylene oxides (e.g., ethylene oxide and propylene        oxide) that are closely related to PEG in their chemistry can be        substituted for PEG in many of its applications.

PAGs are polymers which typically have the properties of solubility inwater and in many organic solvents, lack of toxicity and lack ofimmunogenicity. One use of PAGs is to attach covalently the polymer toinsoluble molecules to make the resulting PAG-molecule “conjugate”soluble. For example, it has been shown that the water-insoluble drugpaclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, etal., J. Org. Chem., 60:331-336 (1995).

Polyalkylated compounds of the invention may typically contain between 1and 500 monomeric units. Other PAG compounds of the invention maycontain between 1 and 200 monomeric units. Still other PAG compounds ofthe invention may contain between 1 and 100 monomeric units. Forexample, the polymer may contain 1, 10, 20, 30, 40, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, or 100 monomeric units. Some compounds of theinvention may contain polymers which include between 5 and 75 or between1 and 50 monomeric units. For example, the polymer may contain 2, 3, 5,6, 7, 8, 10, 11, 12, 13, 15, 16, 17, 18, 20, 25, 30, 33, 34, 35, 40, 45,50, 60, 65, 68, 69, 70, or 75 monomeric units. Preferably, m or n is 7,12, 17, 34 or 69. The polymers may be linear or branched.

It is to be understood that compounds which have been modified by theaddition of a PAG moiety may include a mixture of polymers which have avarying number of monomeric units. Typically, the synthesis of aPAG-modified compound (e.g., a PAG-conjugate) will produce a populationof molecules with a Poisson distribution of the number of monomericunits per polymer in the conjugate. Thus, a compound described as havinga polymer of N=7 monomeric units refers not only to the actual polymersin that population being described as having N=7 monomeric units, butalso to a population of molecules with the peak of the distributionbeing 7. The distribution of monomeric units in a given population canbe determined, e.g., by nuclear magnetic resonance (NMR) or by massspectrometry (MS).

Throughout this application, conventional terminology is used todesignate the isomers as described below and in appropriate text booksknown to those of ordinary skill in the art. (See, e.g., “Principles inBiochemistry”, Lehninger (ed.), page 99-100, Worth Publishers, Inc.(1982) New York, N.Y.; “Organic Chemistry”, Morrison and Boyd, 3rdEdition, Chap. 4, Allyn and Bacon, Inc., Boston, Mass. (1978)).

As described above, certain compounds of the present invention may existin particular geometric or stereoisomeric forms. Except where specifiedto the contrary, the present invention comprehends all such compounds,including cis- and trans-isomers, R— and S-enantiomers, diastereomers,(D)-isomers, (L)-isomers, racemic mixtures thereof and other mixturesthereof as falling within the scope of the invention. Additionalasymmetric carbon atoms may be present in a substituent such as an alkylgroup. All such isomers, as well as mixtures thereof, are included inthis invention.

A carbon atom which contains four different substituents is referred toas a chiral centre. A chiral centre can occur in two different isomericforms. These forms are identical in all physical properties with oneexception, the direction in which they can cause the rotation ofplane-polarized light. These compounds are referred to as being“optically active,” i.e., the compounds can rotate the plane-polarizedlight in one direction or the other.

The four different substituent groups attached to a carbon can occupytwo different arrangements in space. These arrangements are notsuperimposable mirror images of each other and are referred to asoptical isomers, enantiomers or stereoisomers. A solution of onestereoisomer of a given compound will rotate plane polarized light tothe left and is called the levorotatory isomer [designated (−)]; theother stereoisomer for the compound will rotate plane polarized light tothe same extent but to the right and is called dextrorotatory isomer[designated (+)].

The R S system was invented to avoid ambiguities when a compoundcontains two or more chiral centres. In general, the system is designedto rank the four different substituent atoms around an asymmetric carbonatom in order of decreasing atomic number or in order of decreasingvalance density when the smallest or lowest-rank group is pointingdirectly away from the viewer. The different rankings are well known inthe art and are described on page 99 of Lehninger. If the decreasingrank order is seen to be clock-wise, the configuration around the chiralcentre is referred to as R; if the decreasing rank order iscounter-clockwise, the configuration is referred to as S. Each chiralcentre is named accordingly using this system.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, for example the S enantiomer, then it may beprepared by asymmetric synthesis or by derivation with a chiralauxiliary where the resulting diastereomeric mixture is separated andthe auxiliary group cleaved to provide the pure desired enantiomers.Alternatively, where the molecule contains a basic functional group,such as an amino or acidic functional group, such as carboxyl,diastereomeric salts are formed with an appropriate optically-activeacid or base, followed by resolution of the diastereomers thus formed byfractional crystallisation or chromatographic means well known in theart and subsequent recovery of the pure enantiomers.

The compositions and pharmaceutical compositions of the presentinvention may comprise one or more of the compounds of the presentinvention either in a pure form or a partially pure form. Similarly, themethods of the present invention comprise using one or more compounds,wherein the compounds are in a pure form or a partially pure form.

In some embodiments, a composition of the invention may comprise atleast one of the compounds of the present invention, i.e. one or more ofthe compounds represented by the structures of formula I, II, III, IV,A, B, C, D, E, F, A′, B′, C′, D′, E′ and F′. In some embodiments, acomposition of the invention may comprise a mixture of at least two ofthe compounds represented by the structures of formulae I, II, III, IV,A, B, C, D, E, F, A′, B′, C′, D′, E′ and F′. In some embodiments, acomposition of the invention may comprises a mixture of at least five ofthe compounds represented by the structures of formula I, II, III, IV,A, B, C, D, E, F, A′, B′, C′, D′, E′ or F′. In some embodiments, acomposition of the invention may comprise a mixture of at least ten ofthe compounds represented by the structures of formula I, II, III, IV,A, B, C, D, E, F, A′, B′, C′, D′, E′ and F′.

It has been surprisingly found that one or more compounds represented bythe structures of formulae I, II, III, IV, A, B, C, D, E, F, A′, B′, C′,D′, E′ and F′ are effective against AIDS and other viral diseases andagainst HIV+ related infections. Thus, in some embodiments, the presentinvention provides a method for the treatment, prevention or control ofAIDS and other viral diseases and HIV+ related infections in human aswell as in veterinary applications. In some embodiments, said method maycomprise administering to a subject one or more compounds represented bythe structures of formulae I, II, III, IV, A, B, C, D, E, F, A′, B′, C′,D′, E′ and F′. In some embodiments, the method may compriseadministering to a subject a pharmaceutical composition comprising oneor more compounds represented by the structures of formulae I, II, III,IV, A, B, C, D, E, F, A′, B′, C′, D′, E′ and F′.

It has also been found that compounds of the invention should be usefulfor treating AIDS and HIV+ related infections. In experiments describedin the Examples below, compounds of the invention have shown activity invitro. In binding experiments which involved the chemokine receptorCXCR4, compounds of the invention showed activity by preventing thefunction of this receptor, which is the most important receptor for theentrance of the HIV-1 T tropic into its target cell. Treatment of otherconditions in which chemokine receptor inhibition is important ordesirable are also contemplated. For example, control and/or preventionof malignant metastasis is highly important and desirable in cancertreatment. Since the chemokine receptor CXCR4 (and to an extent CXCR3)is involved in cell migration and is possibly the most prominent andimportant receptor in the movement of the malignant cells, compounds ofthe invention, which prevent the function of this receptor, maycontribute to control/prevent the movement of such cells.

Compounds of the invention are also intended for inducing apoptosisand/or inducing AICD. The inventors have found that compounds of theinvention exert an inhibitory effect under certain conditions againstapoptosis, as described in PCT/IB03/04993 cited above. According toPCT/IB03/04993, however, the compounds are administered in a morehydrophilic carrier, e.g., a water-containing carrier. It has now beensurprisingly found that the compounds of the invention, when dissolvedin even a small amount of a carrier which minimizes micellar formationor van der Waals attraction of molecules of the compounds, like DMSO,appear to enhance the development of an immune response, as evidenced byenhanced lymphocyte proliferation and apoptotic effect which arecharacteristic of activation-induced cell death (AICD.) Without wishingto be bound by theory, it is believed that the effect of the DMSO (orother agent which would interfere with micellar formation) is to allowthe compounds to behave differently (i.e., proliferation followed byapoptosis) than when the compounds are in ‘AV micelle’ whereinproliferation is inhibited.

Methods of inhibiting fibrosis or aberrant fibroblast proliferation arealso within the scope of the invention. Preventing fibrosis or aberrantfibroblast proliferation is important in treating or preventing livercirrhosis, for example, and compounds of the invention may exert aninhibitory effect on fibroblast proliferation as shown in the Examples.As such, the compounds of the invention will have usefulness in thisregard.

Methods of administration are well known to a person skilled in the art.Methods of administration include, but are not limited to, parenterally,transdermally, intramuscularly, intravenously, intradermally,intranasally, subcutaneously, intraperitoneally or intraventricularly orrectally. Methods and means of administration are known to those skilledin the art from, for example, U.S. Pat. Nos. 5,693,622; 5,589,466;5,580,859; and 5,566,064, which are hereby incorporated by reference intheir entirety.

In addition, the present invention provides a pharmaceutical compositioncomprising as an active ingredient one or more compounds of the presentinvention, together with one or more pharmaceutically acceptableexcipients. As used herein, “pharmaceutical composition” means atherapeutically effective amount of one or more compounds of the presentinvention together with suitable excipients and/or carriers useful forthe treatment of immuno-allergical diseases, autoimmune diseases andorgan or tissue transplantation rejection. A “therapeutically effectiveamount” as used herein refers to that amount that provides a therapeuticeffect for a given condition and administration regimen. Suchcompositions can be administered by any one of the methods listedhereinabove.

A further aspect of the invention comprehends a compound of theinvention in combination with other compounds of the invention. Acompound of the invention may also be administered in combination withan anti-inflammatory agent, an immunosuppressant, an antiviral agent orthe like. Furthermore, the compounds of the invention may beadministered in combination with a chemotherapeutic agent such as analkylating agent, anti-metabolite, mitotic inhibitor or cytotoxicantibiotic as described above. In general, currently available dosageforms of the known therapeutic agents for use in such combinations willbe suitable.

Combination therapy” (or “co-therapy”) includes the administration of acompound of the invention and at least a second agent as part of aspecific treatment regimen intended to provide the beneficial effectfrom the co-action of these therapeutic agents. The beneficial effect ofthe combination includes, but is not limited to, pharmacokinetic orpharmacodynamic co-action resulting from the combination of therapeuticagents. Administration of these therapeutic agents in combination istypically carried out over a defined time period (usually minutes,hours, days or weeks depending upon the combination selected).“Combination therapy” may, but generally is not, intended to encompassthe administration of two or more of these therapeutic agents as part ofseparate monotherapy regimens that incidentally and arbitrarily resultin the combinations of the present invention.

“Combination therapy” is intended to embrace administration of thesetherapeutic agents in a sequential manner; that is wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents.

Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, oral routes, intravenous routes, intramuscularroutes and direct absorption through mucous membrane tissues. Thetherapeutic agents can be administered by the same route or by differentroutes. For example, a first therapeutic agent of the combinationselected may be administered by intravenous injection while the othertherapeutic agents of the combination may be administered orally.Alternatively, for example, all therapeutic agents may be administeredorally or all therapeutic agents may be administered by intravenousinjection. The sequence in which the therapeutic agents are administeredis not narrowly critical.

“Combination therapy” also can embrace the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients and non-drug therapies (e.g., surgery orradiation treatment.) Where the combination therapy further comprises anon-drug treatment, the non-drug treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and non-drug treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the non-drug treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

The compounds of the invention and the other pharmacologically activeagent may be administered to a patient simultaneously, sequentially orin combination. It will be appreciated that when using a combination ofthe invention, the compound of the invention and the otherpharmacologically active agent may be in the same pharmaceuticallyacceptable carrier and therefore administered simultaneously. They maybe in separate pharmaceutical carriers such as conventional oral dosageforms which are taken simultaneously. The term “combination” furtherrefers to the case where the compounds are provided in separate dosageforms and are administered sequentially.

The compositions and combination therapies of the invention may beadministered in combination with a variety of pharmaceutical excipients,including stabilising agents, carriers and/or encapsulation formulationsas described herein.

In some embodiments, the compositions of the present invention areformulated as oral or parenteral dosage forms, such as uncoated tablets,coated tablets, pills, capsules, powders, granulates, dispersions orsuspensions. In some embodiments, the compositions of the presentinvention are formulated for intravenous administration. In someembodiments, the compounds of the present invention are formulated inointment, cream or gel form for transdermal administration. In someembodiments, the compounds of the present invention are formulated as anaerosol or spray for nasal application. In some embodiments, thecompositions of the present invention are formulated in a liquid dosageform. Examples of suitable liquid dosage forms include solutions orsuspensions in water, pharmaceutically acceptable fats and oils,alcohols or other organic solvents, including esters, emulsions, syrupsor elixirs, solutions and/or suspensions.

Suitable excipients and carriers can be solid or liquid and the type isgenerally chosen based on the type of administration being used.Liposomes may also be used to deliver the composition. Examples ofsuitable solid carriers include lactose, sucrose, gelatin and agar. Oraldosage forms may contain suitable binders, lubricants, diluents,disintegrating agents, colouring agents, flavouring agents,flow-inducing agents, and melting agents. Liquid dosage forms maycontain, for example, suitable solvents, preservatives, emulsifyingagents, suspending agents, diluents, sweeteners, thickeners, and meltingagents Parenteral and intravenous forms should also include minerals andother materials to make them compatible with the type of injection ordelivery system chosen.

This invention is further illustrated in the Examples section whichfollows. This section is set forth to aid in an understanding of theinvention but is not intended to, and should not be construed to, limitin any way the invention as set forth in the claims that followthereafter.

EXAMPLES Example 1 Synthesis of compounds

Compounds of the present invention were synthesised and characterized asdescribed below.

AV 23

0.66g 4 mM 4-hydroxy hydrocinnamic acid and 4 ml thionyl chloride in 30ml cyclohexane were refluxed for 2 hours. Evaporation gave a yellowsolid to which were added 0.9g 4 mM, phenyl alanine ethyl ester HCl, 30ml dichloromethane and 1 ml triethyl amine. After stirring 2 hours atroom temperature, water and KOH were added to neutral pH and thereaction extracted with dichloromethane Evaporation gave a light yellowviscous oil, which was triturated and recrystallized with ethanol togive 0.25 g 18%, white solid,Mp- 213.

NMR CDCl₃ 7.30-6.9(9H,m),4.20(2H,q,J=7.0 Hz),3.30(1H m)3.10(2H,t,J=7.2Hz) 2.90 (2H,m),2.60(2H,t,J=7.2 Hz),1.35(3H,t,J=7.0 Hz).

MS- 341 M⁺,10%),277(15),194(20),165(M-phenethyl ester,100%),149(65) m/e.

AV 24

0.66 g 4 mM 4-hydroxy hydrocinnamic acid and 4 ml thionyl chloride in 30ml cyclohexane were refluxed for 2 hours. Evaporation gave light yellowsolid to which were added 0.5 g 4.1 mM, phenethyl amine, 30 mldichloromethane and 0.6 ml triethyl amine. After stirring for 2 hours atroom temperature, water and KOH were added to neutral pH and thereaction was extracted with dichloromethane. Evaporation gave a viscousoil which was recrystallized with ethanol to give 0.3 g white solid,28%, mp-165.

NMR acetone d₆ 7.35-6.75(9H,m),3.40(2H,q,J=7.1 Hz),2.90(2H,t,J=7.2 Hz)2.75 (2H,t,J=7.2 Hz),2.42(2H,t,J=7.1 Hz). Phenethyl amine-NMR acetone d₆7.2(5H,m),2.96(2H,t,J=7.2 Hz) 2.75 (2H,t,J=7.2 Hz).

MS- 269(M⁺,100%),178(M-benzyl) m/e.

AV 26

0.66 g 4 mM 4-hydroxy hydrocinnamic acid and 4 ml thionyl chloride in 30ml cyclohexane were refluxed for 1.5 hours. Evaporation gave a lightyellow solid to which were added 0.5 g 4.1 mM, histidine amine, 30 mldichloromethane and 0.5 ml triethyl amine. After stirring 2 hours atroom temperature, water and KOH were added to neutral pH and thereaction was extracted with dichloromethane. Evaporation gave a viscousoil which was recrystallized with ethanol to give 0.15 g white solid,15%, mp-245.

NMR acetone d₆ 7.35-(6H,m),3.42(2H,q,J=7.1 Hz),2.93(2H,t,J=7.2 Hz), 2.73(2H,t,J=7.2 Hz),2.45(2H,t,J=7.1 Hz).

MS- 259(M⁺,17%),239(25),213(18),194(100%),185(37) m/e

AV 27

3.2 g DL phenyl alanine, 20 ml ethylene glycol and 7 ml thionyl chloridewere refluxed for 2 hours. Workup as above gave 1.3 g oil which was usedin the synthesis of AV 28.

NMR acetone d₆ 7.35-(5H,m),4.50,3.27,2.90(3H, 12 line ABX), 4.32(2H,t,J=7.0 Hz), 3.76 (2H,t,J=7.0 Hz).

AV 28

1 g 6 mM 4-hydroxy hydrocinnamic acid and 5 ml thionyl chloride in 30 mlcyclohexane were refluxed for 1.5 hours. Evaporation gave a light yellowsolid to which were added 1.2 g AV 27 in 30 ml dichloromethane and 1 mltriethyl amine. After stirring 2 hours at room temperature, water andKOH were added to neutral pH and the reaction was extracted withdichloromethane. Evaporation gave a viscous oil which was recrystallizedwith ethanol to give 0.18 g white solid, 8%,Mp- 224.

NMR acetone d₆ 7.35-6.8(9H,m),3.73-2.50(12H,m).

AV 29

0.22 g 1.3 mM, 4-hydroxy hydrocinnamic acid and 2 ml thionyl chloride in30 ml cyclohexane were refluxed for 1.5 hours. Evaporation gave a lightyellow solid to which were added 0.2 g 1.4 mM, tryptamine in 30 mldichloromethane and 0.3 ml triethyl amine. After stirring 1.5 hours atroom temperature, water and KOH were added to neutral pH and thereaction was extracted with dichloromethane. Evaporation gave a viscousoil which was recrystallized with ethanol to give 0.11 g white solid,27%,mp- 136.

NMR acetone d₆ 7.36(2H,d,J=7.8 Hz),7.0(8H,m),3.48(2H,q,J=7.1Hz),3.05(2H,t,J=7.1 Hz),2.88 (2H,t,J=7.1 Hz),2.52(2H,t,J=7.1 Hz).

AV 30

0.22 g 1.3 mM, 4-hydroxy hydrocinnamic acid and 2 ml thionyl chloride in30 ml cyclohexane were refluxed for 1.5 hours. Evaporation gave lightyellow solid to which were added 0.2 g 1.5 mM, tyramine, 30 mldichloromethane and 0.3 ml triethyl amine. After stirring for 2 hours atroom temperature, water and KOH were added to neutral pH and thereaction was extracted with dichloromethane. Evaporation gave a viscousoil which was recrystallized with ethanol to give 85 mg white solid,23%.

NMR acetone d₆ 7.36(4H,ABq,J=8.8 Hz), 7.20 (4H,Abq,J=8.6 Hz),3.48(2H,q,J=7.1 Hz), 3.05(2H,t,J=7.1 Hz),2.88 (2H,t,J=7.1 Hz),2.52(2H,t,J=7.1 Hz).

AV 32

A. 0.8 g 4-hydroxy cinnamic acid in 40 ml methanol and 10 drops HCl wererefluxed for 12 hours. Workup as above gave 0.6 g oil, 68% yield.

NMR CDCl₃ 7.02,6.75 (4H,Abq,J=8.6 Hz),3.66(3H,s),2.86(2H,t,J=7.4 Hz),2.60 (2H,t,J=7.4 Hz).

B. 0.6 g 3.3 mM, ester from step A and 0.26 g 4.2 mM, ethanol amine wereheated at 100 for 3 hours in an open vessel. Chromatography gave 0.3 grecovered ester followed by amide. The viscous oil was triturated withacetone-methylene chloride and filtered to give 160 mg white solid, 23%yield, Mp- 102.

NMR acetone d₆ 8.10(1H,s,OH),7.03,6.74(4H,Abq,J=8.8 Hz),3.90(1H,t,J=5.2Hz, NH),3.54(2H,q,J=7.1 Hz),3.28(2H,t,J=7.1 Hz), 2.80(2H,t,J=8.2Hz),2.41(2H,t,J=8.2 Hz).

AV 33

0.9g, 6 mM, hydrocinnamic acid and 0.6g 6 equivalents, triphosgen in 30ml dichloromethane and 1.5 ml triethyl amine were stirred 10 minutes atroom temperature and 0.7 g phenethyl amine were added. After 2 hours atroom temperature, workup (HCl) gave a viscous oil which wasrecrystallized with hexane-methylene chloride to give 166 mg whitesolid, 11%, Mp- 91.

NMR acetone d₆ 7.35(10H,m), 3.40(2H,q,J=7.2 Hz), 2.90(2H,t,J=7.4 Hz),2.74 (2H,t,J=7.2 Hz), 2.46(2H,t,J=7.4 Hz).

AV 34

Prepared as AV 33, in the same amount but with tyramine instead ofphenethyl amine. Chromatography and trituration with benzene-hexane gave220 mg white solid, 14%, Mp- 98.

NMR acetone d₆ 7.25(5H,m),6.96,6.75(4H,Abq,J=8.4 Hz),3.43(2H,q,J=6.8Hz),2.94(2H,t,J=7.6 Hz),2.65 (2H,t,J=6.8 Hz),2.42(2H,t,J=7.6 Hz).

AV 35

Prepared as AV 33, 3 mM, from indole propionic acid and tryptamine.Chromatography and trituration with ethanol gave 162 mg white solid,16%, Mp- 142.

NMR acetone d₆ 7.57(2H,d,J=7.8 Hz), 7.36(2H,d,J=7.8 Hz), 7.0(8H,m),3.48(2H,q,J=7.1 Hz), 3.05(2H,t,J=7.1 Hz),2.88 (2H,t,J=7.1 Hz),2.52(2H,t,J=7.1 Hz).

AV 38

Prepared as AV 33, 2 mM, from indole propionic acid and phenethyl amine.Chromatography and trituration with ethanol gave 220 mg white viscousoily solid, 37%.

NMR acetone d₆ 7.57(2H,d,J=7.8 Hz)),7.25-6.97(9H,m),3.44(2H,q,J=7.1 Hz),3.10(2H,t,J=7.1 Hz),2.66(2H,t,J=7.1 Hz),2.51(2H,t,J=7.1 Hz).

AV 43

0.9 g 6 mM hydrocinnamic acid and 0.6 g 6 equivalents, triphosgen in 30ml dichloromethane and 1 ml triethyl amine were stirred for 10 minutesat room temperature and 0.6 g ethanol amine were added. After 2 hours atroom temperature, workup (HCl) gave a viscous oil which wasrecrystallized with hexane to give 124 mg white solid, 11%, Mp- 91.

NMR acetone d₆ 7.30 (5H,m), 3.63(2H,t,J=5.2 Hz), 3.36(2H,q,J=5.2 Hz),2.97(2H,t,J=7.3 Hz), 2.50(2H,t,J=7.3 Hz).

AV 45

Prepared similar to AV 28, but with the triphosgen method, from 6 mMindole propionic acid, AV 27. Chromatography gave 0.35 g viscous oil,13% yield.

NMR CDCl₃ 7.95(1H(br.s), 7.57(2H,d,J=8.0 Hz), 7.36-6.90(9H,m),4.36(2H,t,J=7.1 Hz), 4.17(2H,q,J=7.0 Hz), 3.5-2.8 (7H,m).

0.65 g 3.9 mM, 4-hydroxy hydrocinnamic acid, 15 ml ethylene glycol and 5ml thionyl chloride were refluxed 3 hours. Workup gave 0.5 g 61%, oil.

NMR acetone d₆ 7.02,6.76(4H,Abq,J=8.6 Hz), 4.28(2H,t,J=7.1 Hz),3.63(2H,t,J=7.1 Hz), 2.85, 2.63(4H,m).

AV 48

Prepared as AV 33, 3 mM, from indole propionic acid and tyramine.Chromatography and trituration with ethanol-hexane gave 120 mgpink-white solid, 13%.

NMR acetone d₆ 7.57(2H,d,J=7.8 Hz)), 7.25-6.97(8H,m), 3.44(2H,q,J=7.1Hz), 3.10(2H,t,J=7.1 Hz),2.66(2H,t,J=7.1 Hz),2.51(2H,t,J=7.1 Hz).

AV 49

To 0.7 g 5 mM, hydrocinnamic aldehyde and 0.7 g 5 mM, tyramine in 20 mlethanol was added 0.4 g NaBH₄ and the reaction refluxed 1 hour. Workupgave 0.7 g viscous oil, 55% yield.

NMR acetone d₆ 7.35(5H,m), 7.15, 6.85(4H,Abq,J=8.6 Hz), 2.85(2H,t,J=6.7Hz), 2.70(6H,m),1.80(2H, quin.,J=7.2 Hz).

Example 2 Synthesis of Polyalkylene Glycol Compounds

Polyalkylene glycol compounds were generally synthesised by preparationof the appropriate alcohol compound (e.g., one of the compoundsdescribed in Example 1, or a hydroxylated derivative thereof) and thenconjugation of the alcohol with a polyalkylene glycol (PAG) polymer(e.g., polyethylene glycol (PEG) or polypropylene glycol (PPG)) of thedesired length.

Compound 1, Phenyl Alaninol

1.2 g 32 mM, of LiAlH₄ were added to 2.3 g 10 mM, phenyl alanine ethylester HCl in 50 ml dry ether. After stirring for 2 hours at roomtemperature, water and KOH were added and the reaction product wasextracted with ethyl acetate. After evaporation, 0.8 g of compound 1, alight yellow oil, was obtained.

Compound 1 crystallized on standing. Mp- 70.

NMR CDCl₃ 7.30(5H,m),3.64(1H,dd,J=10.5,3.8 Hz) 3.40(1H,dd,J=10.5,7.2 Hz)3.12 (1H,m),2.81(1H,dd,J=13.2,5.2 Hz),2.52(1H,dd,J=13.2,8.6 Hz)

NMR acetone d₆ 7.30(5H,m),3.76(1H, dt) 3.60(1H,m) 3.30 (1H,t),2.85(2H,m).

Helv. Chim. Acta, 31, 1617(1948). Biels.-E3,Vol. 13, p 1757.

Compound 2, Tyrosinol

To 3 g 12 mM, L-tyrosine ethyl ester HCl in 50 ml dry ether was added1.2 g 32 mM LiAlH₄. After stirring 3 hours at room temperature, waterand KOH were added and the reaction was extracted with ethyl acetate.Evaporation gave 1.1 g of a light yellow oil, 54% yield, which onstanding crystallized. Mp- 85.

NMR CDCl₃ 7.20(4H,AB q,J=8.6 Hz), 3.50(2H,m) 3.20(1H,m), 2.81(2H,m).

NMR tyrosine ethyl ester free base CDCl₃ 7.0,6.56(4H,AB q,J=8.8 Hz),4.20(2H,q,J=7,0 Hz), 3.70,3.0,2.80(3H,12 line ABXm), 1.28.(3H,t,J=7.0Hz).

JACS, 71,305(1949). Biels.-E3,Vol. 13, p 2263.

Compound 3, Tryptophanol

To 3 g 12.9 mM, L-tryptophan methyl ester HCl in 50 ml dry ether wasadded 1.2 gr, 32 mM LiAlH₄. After stirring 6 hours at room temperaturewater and KOH were added and the reaction extracted with ethyl acetate.Evaporation gave 1.23 g light yellow oil, 50% yield. On standingcrystallized. Mp- 65.

NMR CDCl₃ 7.30(5H,m),3.64(1H,dd,J=10.5,3.8 Hz) 3.40(1H,dd,J=10.5,7.2 Hz)3.12 (1H,m),2.81(1H,dd,J=13.2,5.2 Hz),2.52(1H,dd,J=13.2,8.6 Hz)

J. Het. Chem, 13, 777(1976). Biels.-E5, 22 Vol. 12, p 90.

Compound 4, AV 22

0.66 g 4-hydroxy hydrocinnamic acid and 4 ml thionyl chloride in 30 mlcyclohexane were refluxed for 2 hours. After evaporation, a white solidwas obtained, to which 0.65 g oil of Compound 1 (4.3 mM) in 30 mldichloromethane and 0.4 ml triethyl amine were is added. After stirringfor 2 hours at room temperature, water and KOH were added in order toneutralize the pH. The reaction product was extracted withdichloromethane. Evaporation gave 0.8 g of compound 4, light yellowviscous oil. Part of this product was triturated and recrystallized withethanol to give a white solid. Mp- 149.

NMR CDCl₃ 7.30-6.9(9H,m),3.50(2H,m) 3.30(2H,t,J=7.2 Hz) 2.90(3H,m),2.60(2H,t,J=7.2 Hz).

Compound 5, AV 57

0.75 g 5 mM, hydrocinnamic acid and 4 ml thionyl chloride in 30 mlcyclohexane were refluxed for 2 hours. Evaporation gave a white solid towhich were added 0.83 g 5.5 mM, phenyl alaninol in 30 ml dichloromethaneand 0.5 ml triethyl amine. After stirring 3 hours at room temperature,water and KOH were added to neutral pH and the reaction was extractedwith dichloromethane. Evaporation gave 0.57 g of a yellow viscous oil,40% yield.

NMR CDCl₃ 7.40-7.10(10H,m),3.60(2H,m)3.35(2H,t,J=7.2 Hz) 2.95 (3H,m),2.50(2H,t,J=7.2 Hz).

Compound 6, AV 58

0.66 g 4 mM, 4-hydroxy hydrocinnamic acid and 4 ml thionyl chloride in30 ml cyclohexane were refluxed 3 hours. Evaporation gave a light yellowsolid to which were added 0.72 g 4.3 mM, tyrosinol in 30 mldichloromethane and 0.5 ml triethyl amine. After stirring 3 hours atroom temperature water and KOH were added to neutral pH and the reactionwas extracted with dichloromethane. Evaporation gave 0.53 g light yellowviscous oil, 42% yield.

NMR CDCl₃ 7.30,7.20 (8H,2 ABq,J=8.6 Hz),3.40(2H,m) 3.30(2H,t,J=7.2 Hz)2.90 (3H,m),2.60(2H,t,J=7.2 Hz).

Compound 7 AV 59

0.45 g 3 mM, hydrocinnamic acid and 3 ml thionyl chloride in 30 mlcyclohexane were refluxed for 2 hours. Evaporation gave a light yellowsolid to which were added 0.66 g 3.5 mM, tryptophanol in 30 mldichloromethane and 0.4 ml triethyl amine. After stirring 3 hours atroom temperature, water and KOH were added to neutral pH and thereaction was extracted with dichloromethane. Evaporation gave 0.61 gviscous oil, 63% yield.

NMR CDCl₃ 7.50-7.05(10H,m),3.65(2H,m) 3.32(2H,t,J=7.3 Hz) 2.92 (3H,m)2.53(2H,t,J=7.3 Hz).

Compound 8, AV 72

0.45g 3mM, hydrocinnamic acid and 3 ml thionyl chloride in 30 mlcyclohexane were refluxed for 2 hours. Evaporation gave a light yellowsolid to which were added 0.58 g 3.5 mM, tyrosinol in 30 mldichloromethane and 0.4 ml triethyl amine. After stirring for 2.5 hoursat room temperature, water and KOH were added to attain neutral pH andthe reaction was extracted with dichloromethane. Evaporation gave 0.57 glight yellow viscous oil, 63% yield.

NMR CDCl₃ 7.40-7.10(9H,m),3.60(2H,m) 3.35(2H,t,J=7.2 Hz) 2.95(3H,m),2.50(2H,t,J=7.2 Hz).

Compound 9, AV 73

0.38 g 2 mM, 3-indole propionic acid and 2 ml thionyl chloride in 30 mlcyclohexane were refluxed for 2 hours. Evaporation gave light yellowsolid to which were added 0.4 g 2.6 mM, phenyl alaninol in 30 mldichloromethane and 0.3 ml triethyl amine. After stirring 2.5 hours atroom temperature, water and KOH were added to neutral pH and thereaction was extracted with dichloromethane. Evaporation gave 0.47 gpink solid, 75% yield.

NMR CDCl₃ 7.58(1H,d,J=8.0 Hz), 7.40(1H,d,J=8.0 Hz), 7.30-6.9(8H,m),3.50(2H,m) 3.30(2H,t,J=7.5 Hz), 2.95 (3H,m), 2.70(2H,t,J=7.5 Hz).

Compound 10

0.3 g of Compound 4 (AV 22), 0.8 g triphenyl phosphine and 0.55 g ethyldiazo carboxylate were added to 1 g of poly(propylene glycol), (averagemolecular weight ca 1000), in 60 ml dichloromethane. Stirring for 2hours at room temperature, evaporation and chromatography gave 0.65 g ofCompound 10, Formula VII, as a viscous oil.

Additional Compounds Synthesised from Phenyl Alaninol

These compounds include those represented by the following formula:

This compound can also be represented as Formula A, where R is apolypropylene glycol polymer and n is the total number of polypropylenemonomers in the polymer:

AV61:

R=PPG (polypropylene glycol) n=7 MW-706

0.3 g AV 22 (1 mM), 0.8 g 3 mM, triphenyl phosphine and 0.55 g 3.2 mM,ethyl diazo carboxylate were added to 1 g of poly(propylene glycol)(average mol. weight 424, n=7) in 60 ml dichloromethane. After stirringfor 4 hours at room temperature, evaporation and chromatography gave0.55 g viscous oil, a 73% yield.

NMR CDCl₃ 7.30-6.9(9H,m),4.1-3.0(m),2.60(2H,t,J=7.2 Hz),1.2-1.1(m)

AV 62

R=PPG n=12 MW-996

Was prepared as above from 0.2 g AV 22 to give 0.3 g 46% yield.

AV 60

R=PPG n=17 MW-1286

Was prepared as above from 0.1 g AV 22 to give 0.2 g 48% yield.

AV 63

R=PPG n=34 MW-2274

Was prepared as above from 0.1 g AV 22 to give 0.25 g 34% yield.

AV 132

Was prepared as the procedure for AV 72, substituting L(−) tyrosinol forthe (racemic) tyrosinol to give the above compound, and an undeterminedamount of impurity. Use of an adequate protecting scheme to protect theopen phenol ring from attack by the propylene glycol should reduce theamount of impurity.

AV 133

Was prepared as the procedure for AV 22, substituting D(+)phenylalaninol for the (racemic) phenyl alaninol to give the above compound,and an undetermined amount of impurity. Use of an adequate protectingscheme to protect the open phenol ring from attack by the propyleneglycol should reduce the amount of impurity.

AV 134

Was prepared as the procedure for AV 22, substituting L(+)phenylalaninol for the (racemic) phenyl alaninol to give the above compound,and an undetermined amount of impurity. Use of an adequate protectingscheme to protect the open phenol ring from attack by the propyleneglycol should reduce the amount of impurity.

AV 136

R=PPG n=1

Was prepared as above from AV 22 and from AV 133, to give the compoundor its isomer, and an undetermined amount of impurity. Use of anadequate protecting scheme to protect the open phenol ring from attackby the propylene glycol should reduce the amount of impurity.

AV 137

R=PPG n=1

Was prepared as above from AV 22 and from AV 134, to give the compoundor its isomer, and an undetermined amount of impurity. Use of anadequate protecting scheme to protect the open phenol ring from attackby the propylene glycol should reduce the amount of impurity.

Compounds Synthesised from Compound 5, AV 57

AV 86

R=PPG n=7 MW-690

Was prepared as above from 0.22 g AV 57 to give 0.25 g, 47% yield.

AV 87

R=PPG n=17 MW-1270

Was prepared as above from 0.2 g AV 57 to give 0.33 g, 33% yield.

Compounds Synthesised from Compound 9, AV 73

AV 76

R=PPG n=7 MW-729

Was prepared similar to AV 61 above from 0.22 g AV 73 to give 0.23 g,47% yield.

AV 77

R=PPG n=34 MW-2297

Was prepared as above from 0.2 g AV 73 to give 0.35 g, 25% yield.

Compounds Synthesised from Tyrosinol

Compounds Synthesised from Compound 6, AV 58

AV 64

R=PPG n=7 MW-722

Was prepared as above from 0.2 g AV 58 to give 0.21 g, 46% yield.

AV 65

R=PPG n=17 MW-1302

Was prepared as above from 0.23 g AV 58 to give 0.28 g, 29% yield.

Compounds Synthesised from Compound 8, AV 72

AV 74

R=PPG n=7 MW-706

Was prepared similar to AV 61, above, from 0.22 g AV 72 to give 0.26 g50% yield.

AV 75

R=PPG n=34 MW-2274

Was prepared as above from 0.2 g AV 72 to give 0.35 gr, 23% yield.

AV 131

R=PPG n=69 MW-4307

Was prepared as above from AV 72 and poly(propylene glycol (average mol.weight 4,000).

AV 135

R=PPG n=1

Was prepared similar to AV 74, above, from AV 72 and from AV 132, togive the compound or its isomer, and an undetermined amount of impurity.Use of an adequate protecting scheme to protect the open phenol ringfrom attack by the propylene glycol should reduce the amount ofimpurity.

Compounds Synthesised from Tryptophanol

Compounds Synthesised from Compound 7, AV 59

AV 81

R=PPG n=7 MW-729

Was prepared similar to AV 61, above, from 0.22 g AV 59 to give 0.26 g53% yield.

AV 82

R=PPG n=34 MW-2297

Was prepared as above from 0.2 g AV 59 to give 0.35 g 41% yield.

Example 3 Synthesis of(S)2-N(3-O-polypropyleneglycol)propylbenzene)-3-(4-hydroxyphenyl)propylamide(AV 61S, n=7)

Reagents and Instrumentation

1-Hydroxybenzotriazole hydrate (HOBt), Aldrich cat.# 15,726-0;N,N′-dicyclohexylcarbodiimide (DCC), Aldrich cat.# D8-000-2; potassiumcarbonate, Aldrich cat.# 46,781-2; L-phenylalanynol, Fluka cat.# 78100;3-(4-hydroxyphenyl)propionic acid, Fluka cat.# 56190; L-tyrosinolhydrochloride, Aldrich, cat.# 46,999-8; hydrocinnamic acid, Aldrich,cat. # 13,523-2; methanesulfonyl chloride, Aldrich cat.# 47,125-9;poly(propylene glycol) M_(n)=425, Aldrich cat.# 20,230-4; poly(propyleneglycol) M_(n)=1000, Aldrich cat.# 20,232-0. THF and acetonitrile weredried over KOH pellets for at least 48 hours prior to use.methanesulfonyl chloride (mesyl chloride) and pyridine were distilledprior to use. TLC tests were carried out with Merck's 60F₂₅₄ silica-gelon aluminium plates. Column chromatographic separations were made withMerck's Kieselgel 60 silica gel. UV lamp (λ=254 nm) was used to detectUV absorbing spots on the TLC plates. Proton NMR tests were made onBruker's Avance 500 and Avance 200 instruments. Mass-spectral analysesof small molecular weight molecules were made on Bruker's Esquire3000^(plus) mass-spectrometer and of the PPG-containing molecules onBruker's MALDI-TOF (reflex IV) mass-spectrometer. Using a chromatotronis recommended for a better controlled chromatographic separations.

Synthesis of 1 (S isomer)

3-(4-Hydroxyphenyl)-propionic acid (0.2 g, 1.2 mmol), L-phenylalaninol(0.18 g, 1.2 mmol), N-hydroxybenzotriazole (HOBt) (0.16 g, 1.2 mmol) andTHF (5 mL) were put into a round-bottom flask equipped with a magneticstirrer. The flask was cooled in an ice-water bath and a pre-cooledsolution of dicyclohexylcarbodiimide (DCC) (0.26 g, 1.26 mmol) in 3 mLTHF was introduced dropwise into the reaction mixture. The reactionmixture was allowed to stir for additional 1 hour at low temperature andthen for another 2 hours at room temperature. The white precipitateformed was filtered out and the filtrate was evaporated to dryness. Theresidue was dissolved in 10 mL ethyl acetate and the organic phasewashed twice with 1M HCl, then twice with a saturated solution of sodiumbicarbonate solution and then once with water. The organic phase wasdried over anhydrous magnesium sulfate, paper filtered and evaporated toabout a quarter of its original volume. The remaining solution wasallowed to cool and the crystalline precipitate formed was recovered byvacuum filtration to yield 0.24 g of 1 (67%).

Synthesis of 2 (S isomer)

1 (0.1 g, 0.33 mmol), potassium carbonate (0.069 g, 0.5 mmol, thinlycrushed) and THF (3 mL, dried over KOH pellets) were put in around-bottom flask equipped with a magnetic stirrer and a CaCl₂ dryingtube. The mixture was cooled over an ice-salt bath (−10° C.) and apre-cooled solution of di-tert-butyldicarbonate (0.066 g, 0.30 mmole) in2 mL THF (dried) was introduced dropwise. The mixture was allowed tostir at ice temperature for 1 hour and then for 2 days at roomtemperature. The reaction mixture was then evaporated, water (5 mL)introduced and the product was extracted with two 10 mL portions ofethyl acetate. The combined extracts were dried over anhydrous magnesiumsulfate, paper-filtered and the solvent removed. The oily residue wastriturated with a small amount of n-hexane and the solid formedrecovered by vacuum filtration (Yield 0.12 g, 90.1%). Alternatively, theoily residue can be dissolved in an 1:1 mixture of ethyl acetate andhexane and the product recrystallized.

Synthesis of 3 (S isomer)

a. Mesylation of PPG.

106 mg of PPG₄₂₅ (0.25 mmol) was reacted with 90 mole-percent of mesylchloride (26 mg, 2 drops) and 0.4 mmol pyridine (31.6 mg, 2 drops) toafford the mono-mesylated PPG (A). After combining PPG, mesyl chlorideand pyridine, the mesylation reaction was carried out at 0° C. during 30minutes, while stirring, and then continued for another 60 minutes atroom temperature. During mixing the reaction mixture turned fromcolorless to milky-white. The mixture was then dissolved in 5 mLmethylene chloride and the organic phase washed twice with 1M HClsolution, then twice with 1M NaOH solution and once with water. Theorganic phase was dried over anhydrous sodium sulphate, filtered and thesolvent removed.

b. Sodium activation of 2.

0.1 g of 2 (0.25 mmol) was dissolved in 5 mL of absolute ethanol andthen reacted with an equi-molar amount of sodium-ethoxide in absoluteethanol (previously prepared by reacting 0.25 mg-atom of sodium with anaccess of absolute ethanol). The ethanol of the combined solutions wasevaporated to total dryness to yield the sodium salt of 2 (B).

c. Reacting A and B

A was dissolved in 5 mL of a potassium hydroxide-dried acetonitrile andthe solution introduced into a round-bottom flask containing a magneticstirrer. 5 mL of dried acetonitrile solution of B was introduced intothe flask, followed by a catalytic amount (few crystals) of potassiumiodide. A reflux condenser and a gas bubbler adjusted on top of it wereconnected to the reaction vessel and the reaction mixture was allowed toreflux under nitrogen atmosphere, while stirring, during 24 hrs. Thereaction mixture was then paper-filtered and the solvent removed. Theresidue was dissolved in 2 mL of ethyl acetate and then passed through asilica-gel column, using ethyl acetate for elution. The TLC (elutionwith ethyl acetate) UV-absorbing spot at R_(f)=0.55 turned out tocontain the desired product 3 (a mixture of molecules containingdifferent PPG sub-unit lengths), however, containing also some unreactedPPG. Other fractions contained unreacted mesylated PPG anddoubly-mesylated PPG.

Synthesis of 4 (AV 61S)

40 mg of 3 were dissolved in 3 mL of methylene chloride and 10 drops oftri-fluoroacetic acid (TFA) were added. The mixture was gently heated ona hot plate, while at the same time removing the solvent and TFA bydirecting a stream of nitrogen gas at the reaction mixture. Theremaining is an oil-like product, containing target product 4 (a mixtureof molecules containing different lengths of the PPG sub-units and alsounreacted PPG chains).

Example 4 Synthesis of(S)2-N(3-O-(polypropyleneglycol)-1-propyl-4-hydroxybenzene)-3-phenylpropylamide(AV 74S, n=7)

Synthesis of 5 (S isomer)

Hydrocinnamic acid (36.8 mg, 0.24 mmol), L-tyrosinol hydrochloride (0.05g, 0.24 mmol), N-hydroxybenzotriazole (HOBt) (0.033 g, 0.24mmol), sodiumbicarbonate (84 mg, 1 mmol) and THF (5 mL) were put into a round-bottomflask equipped with a magnetic stirrer. The flask was cooled in anice-water bath and a pre-cooled solution of dicyclohexylcarbodiimide(DCC) (53 mg, 0.26 mmol) in 3 mL THF was introduced dropwise into thereaction mixture. The reaction mixture was allowed to stir for another 1hour at low temperature and then 2 hours at room temperature. The whiteprecipitate formed was paper-filtered and the filtrate evaporated todryness. The residue was dissolved in 10 mL ethyl acetate. (Someprecipitate that may occur at this stage must be filtered out). Theclear organic filtrate was twice washed with 1M HCl, then twice with asaturated aqueous solution of sodium bicarbonate and then once withwater. The organic phase was dried over anhydrous magnesium sulfate andpaper filtered. TLC showed three UV-absorbing spots (eluant ethylacetate), one at R_(f)=0.55, the second at R_(f)=0.35 and the third atR_(f)=0.05. Proton NMR (methyl sulfoxide) indicated that the spot atR_(f)=0.35 is of compound 5. This component was separated on asilica-gel column (elution with ethyl acetate), yielding 25 mg of 5(yield: 35%). Alternatively, a quite pure compound 5 (yield 85%) can beobtained by recrystallysing the reaction mixture from methylenechloride.

Synthesis of 6 (S isomer)

Same as synthesis of compound 2. (Yield: 73%).

Synthesis of 7 (S isomer)

Same as synthesis of compound 3. The reaction mixture waschromatographed on a silica-gel column (eluant: ethyl acetate) and afraction containing UV-absorbing spot (R_(f)=0.43, eluant ethyl acetate)proved by MALDI-TOF mass-spectrometer to contain 7 (a mixture ofmolecules containing PPG chains of different size), along with unreactedPPG chains and some unreacted mesylated PPG chains.

Synthesis of 8 (AV 74S)

Same as synthesis of compound 4.

Analogues of compounds 4 (AV 61S) and 8 (AV 74S) containing PPG₁₀₀₀chains (n=17) (AV 60 S and AV 78S, respectively) were prepared using thesame synthetic route as given for the PPG₄₂₅-containing counterparts.

TLC data of the PPG₁₀₀₀ analogues:

PPG₁₀₀₀ analogue of AV 61S (AV 60S): spot at R_(f)=0.40 (eluant: ethylacetate).

PPG₁₀₀₀ analogue of AV 74S (AV 78S): spot at R_(f)=0.43 (eluant: ethylacetate).

Example 5 Effect of AV Compounds on Inhibiting CXCR4 and CXCR3

Shearflow experiments. Soluble, affinity purified seven-domain humanVCAM-1, sVCAM-1 together with the chemokine SDF-1 (ligand for CXCR4) orMig (Ligand for CXCR3) were mixed in coating media (PBS buffered with 20mM sodium bicarbonate pH8.5) and adsorbed as 10 μl drops on apolystyrene plate (60×15 mm Petri dish, Becton Dickinson, Lincoln ParkN.J.) over night at 4° C. The plate was then washed and blocked withhuman serum albumin, HSA (20 mg/ml PBS) for 2 hr at 4° C. Toco-immobilize SDF-1 and Mig with the adhesive substrates, the ligands(VCAM-1) were coated in the presence of active (2 μg/ml) or heatdenatured SDF-1 or Mig and HSA (2 mg/ml) and washed and quenched asabove. A polystyrene plate with coated adhesive substrates was assembledas the lower wall in a parallel plate flow chamber (260 μm gap) mountedon the stage of an inverted phase contrast microscope (Diaphot 300,Nikon) and extensively washed with binding medium. All experiments wereconducted at 37° C. Treated (1 hr incubation with 0.1, 1, 10 μg/ml of AV61, 63, 75, 77) and untreated T cells were diluted with binding mediumand perfused into the chamber at 10⁶ cells/ml by an automated syringepump (Harvard Apparatus, Natick, Mass.). All experiments were recordedon videotape by a long integration camera LIS-700 CCD (Applitech, HolonIsrael) and a SVHS time lapse video recorder (AG-6730 Panasonic). Thehuman T cells were allowed to accumulate for 1 min on the substrate andthen the flow rate was increased to 22 dyn/cm². All recorded images ofcells interacting with the adhesive substrates were analyzed andquantified by computer tracking individual cells. The results obtainedfor each experiment were normalized according to the total number ofvideotaped cells.

As can be seen from the results of the experiments, shown in FIGS. 1A-1C(CXCR4) and FIGS. 2A-2D (CXCR3), compounds of the invention areeffective in inhibiting these cytokine receptors, and as such should beuseful in treating HIV and AIDS via preventing the function of thisreceptor, which is the most important receptor for the entrance of theHIV-1 T tropic into its target cell.

Example 6 Effect of AV Compounds on AICD

T cells were isolated from buffy coats (BC) of consenting normal humandonors (Hadassah Hospital Blood Bank). The BC preparations were diluted1:4 with phosphate-buffered saline (PBS) that contained 10 U/mL heparin.Peripheral blood mononuclear cells were separated by Ficoll/Paquedensity centrifugation. Monocytes and B cells were depleted by plasticadherence and passage through nylon wool columns, respectively. Small Tlymphocytes were harvested from the pellet of a discontinuous Percollgradient. The cells were found to be >80% CD3+ by FACS analysis. Cellswere cultured in the presence of various concentrations of compoundand/or phytohemaglutinin (PHA) (1 μg/ml) T cell mitogen. Proliferationwas measured by culturing 1×10⁵ cells in each well of a 96-wellflat-bottomed microtiter plates. 48 hrs and 7 days following addition ofcompound, 1 μCi ³[H] thymidine was added to each well and the cultureswere incubated for an additional 24 hrs. Samples were harvested andincorporated radioactivity was measured. A 7 day incubation withcompounds led to a significant increase in the proliferation ofPHA-stimulated T cells versus PHA alone.

The effect of AV 63, AV 75, AV 77, AV 131, and polypropylene glycol(“PPG-34”, MW 2000, as a control) on PHA (phytohemagglutinin)-treatedhuman peripheral blood mononuclear cells (PMBCs), using DMSO as acarrier, was investigated. Data from the AV 63 experiment is shown inFIGS. 5B and 6A. Data from the AV 75 experiment is shown in FIGS. 3A, 4Aand 6B. Data from the AV 77 experiment is shown in FIGS. 5A and 6C. Datafrom the PPG-34 experiment is shown in FIGS. 3C and 4B. Data from the AV131 experiment is shown in FIGS. 3B, 3D, 4C and 7A. Data from the AV 77experiment is shown in FIGS. 5A and 6C.

As can be seen from the results of the experiments, shown in theabove-mentioned figures, compounds of the invention surprisingly showthe enhanced lymphocyte proliferation and apoptotic effect which ischaracteristic of activation-induced cell death (AICD.)

Example 7 Effect of AV Compounds on Inhibiting Fibrosis

The effect of AV compounds on fibroblast proliferation was investigated.A thymidine (TdR) incorporation experiment was conducted to determinethe effect of combined AV molecules on human foreskin fibroblast cells(HFF).

Method: Quantitation of [3H]thymidine (TdR) incorporation

Control: Cell culture medium

Inhibitor: >50% decrease of cpm relative to control (drug Doxorubicin)

Procedure:

HFF are cultured in DMEM medium +1% Na-Pyruvate, 1% Pen/Srep, 1% L-Glu,1% none-essential amino acid and 10% FBS. The cells were plated at5×10³/well in a 96 well plate. Extracts were added one day after seedingcells. The cells were incubated for 4 days. 2 days before the end of theexperiment, [3H]-thymidine (1 mCi/well) was added. TdR-1:50 (20 ml in 1ml) (NET-027 Thymidine [methyl-3H] from NEN 6.7 Ci/mmol Batch 3106446)Cells were harvested by adding denaturated agent and scintillationliquid and then counted for 1 min/sample.

Compounds used (AV 61, AV 63 AV 74, AV 75, AV 76, AV 77, AV 81, and AV82) were resuspended in medium, and diluted to the concentrationsindicated in the Figures. Controls were: 1) negative-medium 2)Positive-10^-5M Doxorubicin.

Data from the experiments are shown in FIGS. 8 and 9. As can be seenfrom the results of the experiments, compounds of the invention areeffective inhibitors of fibroblast proliferation.

Example 8 AV Compound Selectivity Index

Anti-HIV-1 results obtained with AV 61 show activity against HIV-1(III_(B) strain) with an EC₅₀ and CC₅₀ of as low as 15.6 μg/ml and 125μg/ml, of pure substance, resulting in a selectivity index of greaterthan 8, and a % PR of as high as 100%, as was determined by anMTT-assay.

The following experimental procedures were employed. MT-4 cells weregrown in RPMI 1640 medium (Life Technologies, Merelbeke, Belgium),supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 2mM L-glutamine, 0.1% sodium bicarbonate and 20 μg /ml gentamicin (LifeTechnologies, Merelbeke, Belgium). The cells were maintained at 37° C.in a humidified atmosphere of 5% CO₂ in air. Every 3-4 days, cells wereseeded at 3×10⁵ cells/ml.

Stocks of HIV-1 III_(B) strain were obtained from the culturesupernatant of 4×10⁵ MT-4 cells/ml infected with HIV at 400 CCID₅₀immediately after complete cytopathic effect (CPE) has appeared. Thevirus titre of the supernatant was determined in MT-4 cells using theReed and Muench end-point dilution method. The virus stocks werealiquoted and stored at −70° C. until used.

Flat-bottom, 96-well plastic microtiter trays (Nunc, Roskilde, Denmark)were filled with 100 ml of complete medium using a Titertek Multidropdispenser (ICN Biomedicals—Flow Laboratories). Stock solution (10× finaltest concentration) of AV 61, were added in 25 μl volumes to two seriesof triplicate wells to allow simultaneous evaluations of their effectson HIV- and mock-infected cells. Serial five-fold dilutions were madedirectly in the microtiter trays using a Biomek 2000 robot (Beckman,Fullerton, Calif.). Untreated control and mock-infected cell sampleswere included. 50 μl of HIV at 100 CCID₅₀ or medium was added to eitherHIV-infected or mock-infected part of a microtiter tray. Exponentiallygrowing MT-4 cells were centrifuged for 5 min at 140×g, and thesupernatants were discarded. The MT4 cells were resuspended at 6×10⁵cells/ml in a flask connected with an autoclavable dispensing cassetteof a Titertek Multidrop dispenser. Under slight magnetic stirring, 50 μlvolumes were then transferred to the microtiter tray wells. The outerrow wells were filled with 200 μl of medium. The cell cultures wereincubated at 37° C. in a humidified atmosphere of 5% CO₂ in air. Thecells remained in contact with the test compounds during the wholeincubation period. Five days after infection, the viability of mock andHIV-infected cells was examined spectrophotometrically by the MTT methodas described hereinbelow.

The MTT assay is based on the reduction of the yellow coloured3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)(Sigma Chemical Co., St. Louis, Mo.) by mitochondrial dehydrogenase ofmetabolically active cells to a blue formazan which can be measuredspectrophotometrically.

To each microtiter well, 20 μl of a solution of MTT (7.5 mg/ml) inphosphate-buffered saline was added using the Titertek Multidrop. Thetrays were further incubated at 37° C. in a 5% CO₂ incubator for 1 hr. Afixed volume of medium (150 μl) was then removed from each cup using theBiomek 2000 robot without disturbing the MT-4 cell clusters containingthe formazan crystals. Solubilization of the formazan crystals wasachieved by adding 100 μl of 10% (v/v) Triton X-100 in acidifiedisopropanol (2 ml concentrated HCl per 500 ml solvent) using the Biomek2000 robot. Complete dissolution of the formazan crystals could beobtained after the trays had been placed on a plate shaker for 10 min(ICN Biomedicals Flow Laboratories). Finally, the absorbances were readin an eight-channel computer controlled Titertek Microplate reader andstacker (Multiskan MCC, ICN Biomedicals—Flow Laboratories) at twowavelengths (540 and 690 nm). The absorbance measured at 690 nm wasautomatically subtracted from the absorbance at 540 nm, to eliminate theeffects of non-specific absorption. Blanking was carried out directly onthe microtiter trays with the first column wells which contained allreagents except MT-4 cells, virus and compounds. All data represent theaverage values for a minimum of three wells. The 50% cytotoxicconcentration (CC₅₀) was defined as the concentration of compound thatreduced the absorbance (OD₅₄₀) of the mock-infected control sample by50%. The percent protection achieved by the compounds in HIV-infectedcells was calculated by the following formula:

$\frac{{( {OD}_{T} ){HIV}} - {( {OD}_{C} ){HIV}}}{{( {OD}_{C} )\mspace{14mu}{mock}} - {( {OD}_{C} ){HIV}}}\mspace{31mu}{expressed}\mspace{14mu}{in}\mspace{14mu}\%$

wherein (OD_(T))HIV is the optical density measured with a givenconcentration of the test compound in HIV-infected cells; (OD_(C))HIV isthe optical density measured for the control untreated HIV-infectedcells; (OD_(C))mock is the optical density measured for the controluntreated mock-infected cells; and all O.D. values were determined at540 nm. The concentration achieving 50% protection according to theabove formula was defined as the 50% effective concentration (EC₅₀).

The results showed that AV 61 has activity against HIV-1 (III_(B)strain) with an EC₅₀ and CC₅₀ of as low as 15.6 μg/ml and 125 μg/ml, ofpure substance, resulting in a selectivity index of greater than 8, anda % PR of as high as 100%, as was determined by an MTT-assay.

Example 9 Effect of AV 61S and PPG7 on PHA-Activated PBMCs

FIG. 10 shows the results of an experiment to investigate the inhibitoryeffect of AV 61S on PHA-activated PBMCs. Human PBMCs were prepared froma blood bank and plated out at a concentration of 10⁵ cells per well ina standard 96-well plate. PHA was added to each well at a concentrationof 10 μg/ml together with AV 61S or PPG-7 (both in PBS) atconcentrations of 50 μg/ml, 1 μg/ml, 100 ng/ml, 1 ng/ml and 100 pg/mlrespectively for AV 61S and 50 μg/ml, 1 μg/ml and 10 ng/ml for PPG-7.The cells were then incubated for 7 days. [3H]-thymidine (NET-027Thymidine [methyl-3H] from NEN 6.7 Ci/mmol Batch 3106446) was added toeach well 48 hours after plating at a concentration of 1 mCi/well; asecond dose was added to each well 18 hours before the end of theexperiment. The cells were then harvested and counted for 1 min./sample.

The controls were medium alone and CsA (1 mg in 1 ml ethanol).

As can be seen in FIG. 10, 60% inhibition relative to the controls wasshown at a concentration of AV 61S of 50 μg/ml.

Example 10 Effect of AV 61S, AV 61 R, AV 74S and AV 74 R onPHA-Activated PBMCs

Example 9 was repeated using separately compounds AV 61S, AV 61R and AV74S and AV 74R. In each case the test compound was diluted in DMSO to afinal concentration of 0.25%. A single dose of [3H]-thymidine (NET-027Thymidine [methyl-3H] from NEN 6.7 Ci/mmol Batch 3106446) was added toeach well 18 hours before the end of the experiment.

FIGS. 11A and 11B show the results for AV 61S and AV 61R and FIGS. 12Aand 12B show the corresponding results for AV 74S and AV 74R. As may beseen, AV 61S shows an inhibitory effect of about 97% relative to thecontrols at a concentration of 100 μg/ml and about 49% at 50 μg/ml. AV61R shows a significantly reduced activity, namely 74% inhibition at 100μg/ml and 42% at 50 μg/ml.

The results for AV 74S and AV 74R show an even more marked differencebetween the S and R enantiomers of the compound. As may be seen fromFIG. 12A, at 100 μg/ml AV 74S shows 100% inhibition relation to thecontrols and 81% at 50 μg/ml. AV 74R, as shown in FIG. 12B, shows noinhibition of activated PBMCs. at 100 μg/ml or less.

Example 11 Toxicity of AV Compounds Against PBMC Cells by Alamar BlueReagent

It has been established in Examples 9 and 10 above that the molecules AV61S and AV 74S have an inhibitory effect on PBMCs (AV 74R had no effectwhereas AV 61R had only a marginal effect) when tested in theconcentrations of 100 μg/ml and 50 μg/ml (61S) and 100 μg/ml; 50 μg/mland 25 μg/ml (74S). A preliminary experiment with Alamar Blue showedthat the inhibitory effect was due to an anti-proliferative activity ofthe molecules rather than to a “killing” effect. In order to check thisin more detail, the present experiment was performed:

Human PBMC cells were obtained from a blood bank and plated out on astandard 96-well plate at a concentration of 10⁵ cells/well. PHA (10μg/ml) and AV 61S (or AV 74S) at different dilutions in DMSO of 100μg/ml, 50 μg/ml, 25 pg/ml, 10 μg/ml and 1 μg/ml were addedsimultaneously to each well. The cells were incubated for 24 hours.

After 24 hours, Alamar Blue reagent was added up to 10% of the totalwell volume.

The controls were medium only and CsA (1 mg/1 ml EtOH).

The contents of each well were then assayed 1 hour, 3 hours and 6 hoursafter addition of Alamar Blue by fluorescence in the manner known tothose skilled in the art to determine the viability of the PBMCs. Theresults are shown in FIGS. 13A-13C.

Example 12 Toxicity of AV Compounds Against PBMC Cells by Alamar BlueReagent

Example 9 was repeated, except that the cells were incubated for 48hours prior to dyeing with Alamar Blue. The results are shown in FIGS.14A-14C.

As shown in FIGS. 13A-13C and FIGS. 14A-14C, Alamar Blue dyeingconfirmed the non-toxic effect of the molecules, except for AV 74S at100 μg/ml which has the same activity and toxicity pattern asCyclosporine (CsA). All other concentrations with AV 61S and AV 74Sshowed an anti-proliferative effect.

Example 13 Toxicity of AV Compounds Against PBMC Cells by Trypan BlueReagent

Examples 11 and 12 were repeated using, in each case, Trypan Bluereagent instead of Alamar Blue. The results, which confirm thoseobtained using Alamar Blue, are shown in FIGS. 15A and 15B respectively.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the invention. Various substitutions,alterations, and modifications may be made to the invention withoutdeparting from the spirit and scope of the invention. Other aspects,advantages, and modifications are within the scope of the invention. Thecontents of all references, issued patents, and published patentapplications cited throughout this application are hereby incorporatedby reference. The appropriate components, processes, and methods ofthose patents, applications and other documents may be selected for theinvention and embodiments thereof.

1. A method of treating an HIV-1 infection comprising administering acomposition comprising a compound of formula II:

wherein:

represents a single or a double bond; R₅ and R_(5′), independently ofeach other, are H, OH or OR₆ where R₆ is a linear or branched C₁-C₄alkyl; Z is —CH₂CH₂O, —CH(CH₃)CH₂O or —CH₂CH(CH₃)O; and m is an integerof 0 or 1, n is an integer of 1-500 and X is O, —CH₂O, —CH₂CH₂O,—CH(CH₃)CH₂O or —CH₂CH(CH₃)O, or m is 1, n is an integer of 0 to 500 andX is —CH₂O, —CH₂CH₂O, —CH(CH₃)CH₂O or —CH₂CH(CH₃)O; or a salt or hydratethereof; in a carrier which minimizes micellar formation or van derWaals attraction of molecules of said compound.
 2. The method of claim1, wherein X is —CH₂O.
 3. The method of claim 1, wherein Z is—CH(CH₃)CH₂O or —CH₂CH(CH₃)O.
 4. The method of claim 1, wherein n is aninteger of 5-75.
 5. The method of claim 4, wherein n is
 69. 6. Themethod of claim 4, wherein n is
 7. 7. The method of claim 4, wherein nis
 17. 8. The method of claim 1, wherein said carrier is DMSO.
 9. Themethod of claim 1, wherein said composition is administered to a patientin need of treatment.
 10. The method of claim 1, wherein at least one ofR₅ or R_(5′) is OH.
 11. The method of claim 4, wherein n is selectedfrom 7, 12, 17, 34, and
 69. 12. The method of claim 1, wherein thecompound is a single enantiomer, further wherein the chiral center atthe position alpha to the amide nitrogen is in the S-configuration. 13.A method of treating an HIV-1 infection comprising administering acomposition comprising a compound selected from

wherein R=PPG (polypropylene glycol) n=7 MW-706 (AV61),

wherein R=PPG n=7 MW-706 (AV74),

(AV135), and

wherein R=PPG n=1 MW-357.44 (AV135 (isomer)) or a salt or hydratethereof; in a carrier which minimizes micellar formation or van derWaals attraction of molecules in said compound.
 14. The method of claim13, wherein said composition is administered to a patient in need oftreatment.
 15. The method of claim 13, wherein said carrier is DMSO. 16.A method of treating an HIV-1 infection comprising administering acomposition comprising AV74S:

wherein * is the S isomer, or a salt or hydrate thereof; in a carrierwhich minimizes micellar formation or van der Waals attraction ofmolecules in said compound.
 17. The method of claim 16, wherein saidcomposition is administered to a patient in need of treatment.
 18. Themethod of claim 16, wherein said carrier is DMSO.
 19. The method ofclaim 1, wherein the compound inhibits CXCR4-mediated adhesion in an invitro assay.
 20. The method of claim 12, wherein X is —CH₂O and m is 1.21. The method of claim 20, wherein

represents a single bond.
 22. The method of claim 21, wherein R₅ is Hand R₅′ is OH.
 23. The method of claim 22, wherein Z is —CH₂CH₂O—. 24.The method of claim 22, wherein Z is —CH(CH₃)CH₂O— or —CH₂CH(CH₃)O. 25.The method of claim 23, wherein n is 1, 2, or
 7. 26. The method of claim24, wherein n is 1, 2, or
 7. 27. The method of claim 25, wherein n is 1.28. The method of claim 26, wherein n is 1.